Developing a laboratory cultivation protocol for local species of Porphyra spp. Andreas Quale Lavik MSc in Biology Submission date: May 2016 Supervisor: Kjell Inge Reitan, IBI Co-supervisor: Silje Forbord, SINTEF Norwegian University of Science and Technology Department of Biology Acknowledgements This thesis was written at the Department of Biology, NTNU, Trondheim 2016. All experiments were conducted at Sintef SeaLab and NTNU Centre of Fisheries and Aquaculture. The thesis was part of the project "Dyrking og foredling av makroalger av høy kvalitet til nye matprodukter", financed by the Regional Research Funds Vestlandet (project number: 245326) and Skattefunn (project number: 247546). The project is owned by Austevoll Seaweed Farm AS, and other partners are SINTEF Fisheries and Aquaculture (project leader), NTNU and Ørjan Johannesen. The thesis was written under supervision of Kjell Inge Reitan, NTNU, and Silje Forbord, SINTEF. First I would like to thank my supervisors, Kjell Inge Reitan and Silje Forbord, for outstanding help, input, supervision and support. It could not have been done without, and your guidance and patience has been of crucial importance. I would also like to thank Tora Bardal for assistance in the laboratory, and for helpful tips, tricks and guidance in the use of equipment and software. I would also like to thank Dag Altin, for fruitful discussions and helpful suggestions on methodology and equipment use. Also a thank you goes to Andreas Hagemann at SINTEF, for guidance and friendship. Another thank you goes to my fellow students at NTNU SeaLab, for creating a great environment, you are truly all outstanding people whom will be greatly missed. Also a special nod to Anna Rongved for support, encouragement and friendship, as well as her role as LAKS leader through the past year. Good job! Trondheim, May 2016 Andreas Quale Lavik 1 Abstract The cultivation of Porphyra for use in food has a long tradition in Asian countries, and with recent food trends and nutritional awareness, macroalgae has become a popular ingredient throughout the rest of the world as well. The Norwegian coast has several species of Porphyra suited for use in many ways, but harvesting wild material is time consuming and limited by few occurrences in the wild. Cultivation is thus the preferred option for future exploitation of the genus. This study is aimed at adapting existing cultivation techniques to local species and environmental factors, for indoor cultivation. A field search was performed to map local occurrences of the genus in the Trondheimsfjord, and a laboratory protocol was established for cultivation of collected material. Several variables were applied at different stages of the life cycle; stress for induction of spore release, treatment with germanium dioxide for decontamination, variations in light intensities and application of scallop shell as a substrate for growth. Also a method for quantification of conchochelis (sporophyte stage) growth were developed and applied. The field search found that the genus is scarcely represented in local waters, with few and far between occurrences. The laboratory experiment proved that contaminants like diatoms is a challenge, but manageable with correct treatment. Tolerance levels for germanium dioxide were also established, and the experiment showed that the local species of Porphyra are well suited for indoor cultivation under the given conditions. The experiment was concluded when the life cycle was fulfilled, from wild collected gametophytes, through the conchochelis sporophyte stage, to growing gametophytes in the laboratory. 2 Sammendrag Dyrking av Porphyra til bruk i mat har en lang tradisjon i Asiatiske land, og med nye trender og bevissthet rundt næringsinnhold har makroalger blitt en populær ingrediens i resten av verden og. Norskekysten har flere arter Porphyra som er egnet for mange typer bruk, men høsting av frittvoksende materiale er tidskonsumerende og begrenset av få forekomster. Dyrkning er dermed den foretrukne metoden for videre utnyttelse av slekten. Denne studien har som mål å tilpasse eksisterende dyrkningsteknikker til lokale arter og miljømessige faktorer, for innendørs dyrkning. Ett feltsøk ble gjennomført for å kartlegge lokale forekomster av slekten i Trondheimsfjorden, og en laboratoreprotokoll ble etablert for dyrkning av innsamlet materiale. Flere variabler ble anvendt i forskjellige stadier av livssyklusen; stress for å indusere sporeslipp, behandling med germanium dioxide for dekontaminering, variasjoner i lysintensitet og applisering av kamskjell som vekstsubstrat. I tillegg ble en metode for kvantifisering av conchochelis (sporofytt stadiet) vekst utviklet og anvendt. Feltsøket fant at slekten er knapt representert i lokale farvann, med få forekomster med stor avstand i mellom. Laboratorieforsøket bevist at kontaminering av diatomer er en utfordring, men håndterbar med korrekt behandling. Toleransenivåer for germanium dioxide ble og etablert, og forsøket viste at lokale den lokale arten av Porphyra er godt egnet for innendørs kultivering under de gitte forhold. Forsøket endte da livssyklusen var fullført, fra frittvoksende gametofytter, gjennom conchochelis sporofytt stadiet, til voksende gametofytter i laboratoriet. 3 Table of contents Acknowledgements ................................................................................................................................. 1 Abstract ................................................................................................................................................... 2 Sammendrag ........................................................................................................................................... 3 Table of contents ..................................................................................................................................... 4 Glossary ................................................................................................................................................... 6 1 Introduction .......................................................................................................................................... 7 1.1 Taxonomy and distribution ........................................................................................................... 7 1.1.1 Taxonomy ............................................................................................................................... 7 1.2.2 Distribution ............................................................................................................................. 8 1.2 Morphology and life cycle ............................................................................................................. 9 1.2.1 Morphology ............................................................................................................................ 9 1.2.2 Life cycle ............................................................................................................................... 10 1.3 Cultivation of Porphyra spp. ........................................................................................................ 12 1.3.2 Porphyra aquaculture ........................................................................................................... 12 1.3.3 Nutritional value and uses for human consumption ............................................................ 13 1.4 Study aims and approach ............................................................................................................ 14 2 Materials and methods ...................................................................................................................... 15 2.2 Field search for natural occurrences of Porphyra spp. in the Trondheimsfjord. ........................ 15 2.3 Collection of material .................................................................................................................. 16 2.4 Spore isolation ............................................................................................................................. 19 2.5 Laboratory cultivation of Porphyra spp. life cycle ....................................................................... 21 2.5.1 Variables for growth phases ................................................................................................. 25 2.6 Conchosporangium quantification .............................................................................................. 30 2.7 Decontamination – NaOCl, SSW washing and air drying ............................................................ 30 2.9 Statistics....................................................................................................................................... 31 3 Results ................................................................................................................................................ 32 3.2 Field search for natural occurrences of Porphyra spp. in the Trondheimsfjord. ........................ 32 3.3 Collected material ....................................................................................................................... 33 3.4 Spore isolation ............................................................................................................................. 38 3.5 Laboratory cultivation of Porphyra spp. and growth variables ................................................... 39 3.5.1 Induction of carpospore release and effects